Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have analyzed the transcription of a cloned silkworm tRNA2Ala gene in germinal vesicle extracts of X. laevis oocytes. The primary transcript was sequenced; it is 98 nucleotides long, beginning with a 5' triphosphate nucleotide and ending in a 3' oligouridine stretch. After transcription for long periods of time, enzymes in the frog extract also process the tRNA2Ala precursor to remove extra 5' and 3' nucleotides and to add a CCA end. The twenty-two extra nucleotides at the 3' end of this precursor are recovered as an intact fragment, implicating a new site of endoribonuclease cleavage in eucaryotic tRNA processing. This enzyme activity has also been demonstrated by reincubation of isolated pre-tRNA2Ala with a germinal vesicle extract. The products of in vitro cleavage are the same as those seen in the transcription reactions. The tRNA2Ala precursor molecules are made faithfully in the system with as few as 6 bp of Bombyx morti DNA upstream of the transcription initiation site of the tRNA2Ala gene. This result narrows down the minimal amount of DNA adjacent to the 5' end of a eucaryotic tRNA gene needed to support proper initiation by RNA polymerase III.
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PMID:Transcription of a cloned Bombyx mori tRNA2Ala gene: nucleotide sequence of the tRNA precursor and its processing in vitro. 26 Jun 97

The tRNATyr precursor molecule, synthesized from phi 80 psu3+ DNA (containing a single tRNA gene) by DNA-dependent RNA polymerase and q factor, was about 205 nucleotides long. The main product of its digestion with a ribonuclease tii preparation from Escherichia coli showed the same electrophoretic mobility as tRNAtyr precursor isolated in vivo and was found to be identical to it when analysed using fingerprint techniques. This intermediate precursor synthesized in vitro was converted further by processing with ribonuclease P into an RNA identical size to mature tRNATyr. It was concluded that the initiation of transcription of the tRNATyr gene in vitro occurs at the same site as that of transcription in vivo and a termination occurs at about 80 nucleotides beyond the CCA end of tRNATyr.
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PMID:Processing by ribonuclease II of the tRNATyr precursor of Escherichia coli synthesized in vitro. 32 7

Structural resemblance of the human Alu family with a subset of vertebrate tRNAs was detected. Of four tRNAs, tRNA(Lys), tRNA(Ile), tRNA(Thr), and tRNA(Tyr), which comprise a structurally related family, tRNA(Lys) is the most similar to the human Alu family. Of the 76 nucleotides in lysine tRNA (including the CCA tail), 47 are similar to the human Alu family (60% identity). The secondary structure of the human Alu family corresponding to the D-stem and anticodon stem regions of the tRNA appears to be very stable. The 7SL RNA, which is a progenitor of the human Alu family, is less similar to lysine tRNA (55% identity), and the secondary structure of the 7SL RNA folded like a tRNA is less stable than that of the human Alu family folded likewise. Insertion of the tetranucleotide GAGA, which is an important region of the second promoter for RNA polymerase III in the Alu sequence, occurred during the deletion and ligation process to generate the Alu sequence from the parental 7SL RNA. These results suggest that the human Alu family was generated from the 7SL RNA by deletion, insertion, and mutations, which thus modified the ancestral 7SL sequence so that it could form a structure more closely resembling lysine tRNA. The similarities of several short interspersed sequences to the lysine tRNA were also examined. The Galago type 2 family, which was reported to be derived from a methionine initiator tRNA, was also found to be similar to the lysine tRNA. Thus lysine tRNA-like structures are widespread in genomes in the animal kingdom. The implications of these findings in relation to the mechanism of generation of the human Alu family and its possible functions are discussed.
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PMID:Transfer RNA-like structure of the human Alu family: implications of its generation mechanism and possible functions. 170 38

The cleavage specificities of the RNase P holoenzymes from Escherichia coli and the yeast Schizosaccharomyces pombe and of the catalytic M1 RNA from E. coli were analyzed in 5'-processing experiments using a yeast serine pre-tRNA with mutations in both flanking sequences. The template DNAs were obtained by enzymatic reactions in vitro and transcribed with phage SP6 or T7 RNA polymerase. The various mutations did not alter the cleavage specificity of the yeast RNase P holoenzyme; cleavage always occurred predominantly at position G + 1, generating the typical seven base-pair acceptor stem. In contrast, the specificity of the prokaryotic RNase P activities, i.e. the catalytic M1 RNA and the RNase P holoenzyme from E. coli, was influenced by some of the mutated pre-tRNA substrates, which resulted in an unusual cleavage pattern, generating extended acceptor stems. The bases G - 1 and C + 73, forming the eighth base pair in these extended acceptor stems, were an important motif in promoting the unusual cleavage pattern. It was found only in some natural pre-tRNAs, including tRNA(SeCys) from E. coli, and tRNAs(His) from bacteria and chloroplasts. Also, the corresponding mature tRNAs in vivo contain an eight base pair acceptor stem. The presence of the CCA sequence at the 3' end of the tRNA moiety is known to enhance the cleavage efficiency with the catalytic M1 RNA. Surprisingly, the presence or absence of this sequence in two of our substrate mutants drastically altered the cleavage specificity of M1 RNA and of the E. coli holoenzyme, respectively. Possible reasons for the different cleavage specificities of the enzymes, the influence of sequence alterations and the importance of stacking forces in the acceptor stems are discussed.
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PMID:Sequence changes in both flanking sequences of a pre-tRNA influence the cleavage specificity of RNase P. 170 37

A gene (rtr-1) coding for the tRNAArgACG has been isolated and characterized from the nematode, Caenorhabditis elegans. The coding portion is not interrupted by an intron and is followed by a track of four thymidines associated with termination by RNA polymerase III. The predicted mature product is 76 nucleotides (nt) long including the CCA tail, and is specific for the most used Arg codon in C. elegans. The gene can be transcribed and processed in a homologous in vitro system. The 82-nt primary transcript begins at the first purine upstream from the mature tRNA 5' end and terminates after the first thymidine of the terminator signal.
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PMID:Cloning and expression in vitro of a gene encoding tRNAArgACG from the nematode Caenorhabditis elegans. 199 92

To study the mechanism involved in the 3'-processing of mitochondrial tRNA precursors, we examined tRNA processing in a reconstituted system with a yeast mitochondrial extract. Two mitochondrial tRNA(Glu) precursors synthesized from SP6 RNA polymerase-directed transcription system were used as substrates. One contained a 214-nucleotide 5' terminus and 115-123-nucleotide 3' trailer. The other had the same sized 3' trailer, but contained a mature 5' terminus. An endonucleolytic activity was identified in a mitochondrial S30 fraction which cleaves the 3' terminus of the latter tRNA precursor precisely at the in vivo CCA addition site. No cleavage of the 5'-extended precursor was observed in vitro. This mitochondrial 3'-processing activity was partially purified using DEAE-CL-6B chromatography. It removes the 3' trailer sequence from the 5'-matured precursor leaving a 3'-hydroxyl group on the processed tRNA and a 5'-phosphate group on the trailer. The resulting tRNA product serves as a substrate for tRNA nucleotidyltransferase which catalyzes the addition of CCA residues to the tRNA to complete its 3' maturation. Thus, yeast mitochondrial 3'-tRNA processing events resemble those found in eucaryotic cytoplasmic/nuclear systems where a single endonucleolytic cleavage is responsible for the formation of the 3' end of the tRNAs. This is in contrast to the multistep 3'-processing events known to occur in procaryotes.
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PMID:Biosynthesis of tRNA in yeast mitochondria. An endonuclease is responsible for the 3'-processing of tRNA precursors. 284 29

A phage lambda clone containing a 13.1-kb human DNA fragment was isolated and found to contain a tRNA gene encoding a glycine tRNA. The nucleotide sequence of the gene and its flanking regions has been determined. The gene does not have an intervening sequence nor does it encode the 3'-terminal CCA sequence found in mature tRNAs. Although this tRNA gene has an anticodon sequence of CCC, it has a striking homology (96%) with a human glycine tRNA which has an anticodon of GCC. As in other eukaryotic tRNA genes, the coding region contains a characteristic internal split promoter sequence, and the 3'-flanking region has a typical RNA polymerase III termination site of five consecutive T residues. There is no apparent sequence in the 5'-flanking region which could serve as a regulatory element. This gene is accurately transcribed in vitro by RNA polymerase III using a HeLa cell-free system. During the course of in vitro transcription, larger precursor tRNAGlyCCC transcripts are processed to yield a mature-sized tRNA product. A precursor-product relationship was established by comparing the ribonuclease A fingerprints of the precursor and product tRNA transcripts.
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PMID:Nucleotide sequence and transcription of a gene encoding human tRNAGlyCCC. 298 90

The nucleotide sequence (25,320 base-pairs) of a part of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha was determined. This region encodes putative genes for four tRNAs, isoleucine tRNA(CAU), arginine tRNA(CCG), proline tRNA(UGG) and tryptophan tRNA(CCA); eight photosynthetic polypeptides, the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL), 51,000 Mr photosystem II chlorophyll alpha apoprotein (psbB), apocytochrome b-559 polypeptides (psbE and psbF), 10,000 Mr phosphoprotein (psbH), cytochrome f preprotein (petA), cytochrome b6 polypeptide (petB), and cytochrome b6/f complex subunit 4 polypeptide (petD); 13 ribosomal proteins (L2, L14, L16, L20, L22, L23, L33, S3, S8, S11, S12, S18 and S19); initiation factor 1 (infA); ribosome-associating polypeptide (secX); and alpha subunit of RNA polymerase (rpoA). Functionally related genes were located in several clusters in this region of the genome. There were two ribosomal protein gene clusters: rpl23-rpl2-rps19-rpl22-rps3-rpl16-+ ++rpl14-rps8-infA-secX-rps11-rpoA, with a gene arrangement similar to that of the Escherichia coli S10-spc-alpha operons, and the rps12'-rpl20-rps18-rpl33 cluster. There were gene clusters encoding photosynthesis components such as the psbB-psbH-petB-petD and the psbE-psbF clusters. Thirteen open reading frames, ranging in length from 31 to 434 amino acid residues, remain to be identified.
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PMID:Structure and organization of Marchantia polymorpha chloroplast genome. III. Gene organization of the large single copy region from rbcL to trnI(CAU). 319 36

A bacteriophage lambda clone containing a 20-kb human DNA segment was isolated and found to harbor a cluster of four tRNA genes. An 8.2-kb HindIII subfragment encompassing the genes was cloned into pBR322 for restriction mapping and DNA sequence analysis. The genes were found to be arranged as two tandem pairs, separated by 3 kb. A proline tRNAAGG gene is separated from a leucine tRNAAAG gene by a 724-bp intergenic region in the first pair, and a second proline tRNAAGG gene is 316 bp from a threonine tRNAUGU gene in the second pair, with the leucine tRNA gene being of opposite polarity to the other three genes. A putative Alu-like element was found to occur within a 2.0-kb DNA fragment, at least 0.7 kb from the tRNA gene cluster. The coding sequences of the two proline tRNAAGG genes are identical. The coding regions of all four tRNA genes contain consensus internal split promoter sequences and do not have intervening sequences nor the CCA trinucleotide found in mature tRNAs. The 3'-flanking regions of these four tRNA genes have normal RNA polymerase III termination sites of at least four consecutive T nucleotides. No apparent homologies occur between the 5'-flanking regions of these genes. All four tRNA genes are accurately transcribed in an in vitro HeLa cell-free system, and the RNase T1 fingerprints of the mature-sized tRNA transcripts were found to be consistent with the DNA sequences of the genes.
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PMID:Nucleotide sequence and transcription of a human tRNA gene cluster with four genes. 355 25

Using molecular cloning techniques, a clone containing a 7-kb insert of Spiroplasma species BC3 DNA that hybridized strongly to total labeled Spiroplasma tRNA was identified. Sequence analysis of a portion of this recombinant plasmid identified a cluster of tRNA genes. The gene order was as follows: tRNACys-tRNAArg-tRNAPro-tRNAAla-tRNAMet-tRNAIle and a portion of tRNASer All the genes encode the 3'-terminal CCA and have very high A + T and relatively long intergenic regions. An RNA polymerase promoter site was found upstream of the tRNACys gene. The tRNA gene cluster found in Spiroplasma can be compared with a similar Bacillus subtilis gene cluster, which raises interesting questions concerning gene organization and transcription.
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PMID:A Spiroplasma tRNA gene cluster. 609 3


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